JP6253210B1 - Method for preventing or treating swine epidemic diarrhea, vaccine, and vaccine kit - Google Patents

Abstract

[PROBLEMS] To prevent and treat swine epidemic diarrhea that is excellent in inducing activity of neutralizing antibody specific to swine epidemic diarrhea virus and inducing activity of humoral immune response and can efficiently prevent or treat swine epidemic diarrhea. To provide a preventive or therapeutic method, a vaccine kit, a vaccine for oral or nasal administration, and a vaccine for intramuscular administration. A first administration step of administering a swine epidemic diarrhea virus live vaccine and an adjuvant to a pig by either oral administration or nasal administration; and the porcine epidemic diarrhea virus inactivated vaccine and adjuvant; Is a method for preventing or treating swine epidemic diarrhea, comprising a second administration step of administering to a pig by intramuscular administration. [Selection figure] None

Description

  The present invention relates to a method for preventing or treating swine epidemic diarrhea, a vaccine for oral or nasal administration, a vaccine for intramuscular administration, and a vaccine kit.

  Porcine epidemic diarrhea (PED) is caused by oral infection with PED virus (PEDV) contained in the feces of infected pigs, and is mainly characterized by watery diarrhea and anorexia. It is a viral infectious disease showing very high mortality in the following suckling pigs (see Non-Patent Document 1). Once PEDV enters a pig farm, it is difficult to clean, which is problematic in that it causes enormous economic damage. In fact, swine epidemic diarrhea has recently spread in North America, mainly Asian countries and the United States, resulting in tremendous economic damage (see Non-Patent Document 2).

  The target cells of PEDV are epithelial cells of the digestive tract, particularly the jejunum and ileum (see Non-Patent Document 3). Some PEDV are thought to enter the bloodstream through submucosal lymphoid tissues, and there are times when the PEDV gene is detected in systemic organs. However, PEDV does not show secondary growth in other organs, and after it grows in gastrointestinal epithelial cells, it is excreted from feces and becomes a new source of infection. In pigs after the weaning period, the sensitivity to PEDV decreases, and even if PEDV is forcibly administered orally, it may be asymptomatic. However, when pigs under stress such as infection with other pathogens, childbirth, and overcrowding are exposed to PEDV, symptoms such as loss of appetite and diarrhea may appear transiently (non-patented). Reference 4).

  A vaccine against swine epidemic diarrhea (hereinafter sometimes referred to as “PED vaccine”) has been marketed in Japan, Korea, China, the Philippines, and the United States since the late 1990s. These conventional PED vaccines are inoculated during the gestation period of mother pigs, secrete neutralizing antibodies against PEDV in the milk of postpartum mother pigs, and the suckling pigs ingest the milk to ingest the milk. It has been designed based on the mechanism of neutralizing PEDV that has entered the digestive tract (see Non-Patent Document 5). A suckling pig cannot escape from PEDV infection only by a transfer antibody contained in colostrum, and continues to ingest milk containing a neutralizing antibody against PEDV at a high concentration by the PED vaccine throughout the feeding period. It can neutralize, thereby preventing the development of swine epidemic diarrhea and reducing symptoms.

  However, if the piglet falls into a situation where it cannot take milk for some reason, the piglet is infected with PEDV and becomes serious. In addition, when PEDV antibody-negative sows and low-immune sows are exposed to a large amount of PEDV, they have systemic symptoms accompanied by a decrease in milk production, and the PED vaccine cannot be fully effective. It was.

  Furthermore, the effects of the conventional PED vaccine are limited to the reduction of symptoms caused by PEDV and the reduction of mortality in piglets, and the induction of the humoral immune response is insufficient and the response of IgA antibody is sufficiently induced. There was also a problem that it was not possible (see Non-Patent Document 6).

  Therefore, a new vaccine against swine epidemic diarrhea that can not only induce the production of PEDV-specific neutralizing antibodies in milk but also induce an immune response that can protect against infection in the mother pig itself, At present, it is strongly desired to provide a method for preventing or treating epidemic diarrhea.

Zimmerman J.M. J. et al. et al. , Diseases of sine, 10th edition, Wiley-Blackwell 2012 Lee C. , Virol J, 2015, Vol. 12, p. 193 Sueoshi M. et al. et al. , J. et al. Comp. Pathol, 1995, Vol. 113 (1), pp. 59-67 Miyasaki Ayako, “Pig epidemic diarrhea (PED), which has been prevalent in recent years”, Veterinary epidemiology magazine, 2014, 18 (1), pp. 85-89 Tetsuro Sato, “Characteristics and Effects of Live Porcine Diarrhea Vaccine”, Monthly Pig Journal, February 2014, pp. 22-25 Song D. et al. , Clin. Exp. Vaccine Res. , 2015, Vol. 4 (2), pp. 166-176

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is excellent in the activity of inducing the production of neutralizing antibodies specific for swine epidemic diarrhea virus and the activity of inducing humoral immune response, and can effectively prevent or treat swine epidemic diarrhea. It is an object of the present invention to provide a method for preventing or treating diarrhea, a vaccine kit, a vaccine for oral or nasal administration, and a vaccine for intramuscular administration.

  As a result of intensive studies to achieve the above object, the present inventors have administered a live vaccine and an adjuvant of swine epidemic diarrhea virus to pigs by either oral administration or nasal administration, Intramuscular addition of an inactivated diarrhea virus vaccine and an adjuvant enhances the induction of neutralizing antibodies specific for porcine epidemic diarrhea virus compared to vaccines against conventional porcine epidemic diarrhea It was also found that IgA antibodies specific for swine epidemic diarrhea virus can also be induced. Furthermore, it has been found that these antibodies are secreted into the milk and not only enable infection protection in the piglets, but also can protect the infection of the mother pigs themselves.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> a first administration step in which a swine epidemic diarrhea virus live vaccine and an adjuvant are administered to pigs by either oral administration or nasal administration;
A method for preventing or treating porcine epidemic diarrhea, comprising: a second administration step of administering the inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant to the pig by intramuscular administration.
<2> Porcine epidemic diarrhea virus live vaccine for oral or nasal administration, adjuvant for oral or nasal administration, inactivated vaccine of porcine epidemic diarrhea virus for intramuscular administration, and adjuvant for intramuscular administration It is a vaccine kit characterized by having.
<3> Used for pigs given booster immunity after intramuscular administration of an inactivated vaccine and an adjuvant of swine epidemic diarrhea virus,
It is a vaccine for oral or nasal administration, characterized in that it comprises a live vaccine of the porcine epidemic diarrhea virus and an adjuvant.
<4> Used for pigs that were given the initial immunization by oral administration or nasal administration of live vaccine and adjuvant of porcine epidemic diarrhea virus,
A vaccine for intramuscular administration, comprising the inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant.

  According to the present invention, the conventional problems can be solved and the object can be achieved, and the neutralizing antibody production-inducing activity specific to swine epidemic diarrhea virus and the humoral immune response-inducing activity are excellent. It is possible to provide a method for preventing or treating porcine epidemic diarrhea, a vaccine kit, a vaccine for oral or nasal administration, and a vaccine for intramuscular administration that can efficiently prevent or treat porcine epidemic diarrhea.

FIG. 1 is a diagram showing the results of phylogenetic tree analysis of the PEDV spike gene.

(Prevention or treatment of swine epidemic diarrhea)
The method for preventing or treating swine epidemic diarrhea of the present invention includes a first administration step and a second administration step, and further includes other steps as necessary.

<First administration step>
The first administration step is a step of administering a live porcine epidemic diarrhea virus vaccine and an adjuvant to pigs by either oral administration or nasal administration.

<< Live porcine epidemic diarrhea virus (PEDV) vaccine >>
The live PEDV vaccine is a vaccine that attenuates the toxicity of PEDV.
PEDV is a virus belonging to the family Coronaviridae and Alphacoronavirus , and is classified into two genetic groups, Group I and Group II.

  There is no restriction | limiting in particular as classification of PEDV used for the said live vaccine of PEDV, According to the objective, it can select suitably, Either of the said group I and the said group II may be sufficient. These PEDV may be used individually by 1 type, and may use 2 or more types together.

  There is no restriction | limiting in particular as an acquisition method of the said PEDV, According to the objective, it can select suitably, For example, the method of isolate | separating and obtaining from the pig infected with PEDV, the method of using a commercial item, etc. are mentioned.

The following method etc. are mentioned as a specific example of the method of isolate | separating and obtaining from the pig infected with the said PEDV.
After collecting the small intestine from a pig that developed porcine epidemic diarrhea (PED) and preparing a tissue emulsion, the tissue emulsion was orally administered to a pig not infected with PED, and after several days, the small intestine of the pig was collected. Using this, a tissue emulsion is prepared. After appropriately selecting such passages, trypsin was added to the tissue emulsion obtained from the last passage so that the final concentration was 5 μg / mL to 10 μg / mL, and the confluent Vero cells were added. Inoculate and incubate in 5 μg / mL to 10 μg / mL trypsin culture. The cytopathic effect (CPE) is confirmed daily with an optical microscope, and the culture is stopped when CPE is confirmed. RNA is extracted from the culture supernatant of Vero cells in which CPE has been confirmed by a conventional method, and RT-PCR is performed using the primer sequences represented by SEQ ID NOs: 1 to 16. Then, the amplified RT-PCR product is cloned into an arbitrary vector by a conventional method to obtain a plasmid. The base sequence of the PEDV spike gene (hereinafter sometimes referred to as “PEDV S gene”) in this plasmid was analyzed by a conventional method, and each analyzed sequence fragment was assembled. For example, MEGA 4.0 software (http: /// /Www.megasoftware.net), it is possible to determine which of the group I and group II PEDV belongs to the genetic group by performing a phylogenetic tree analysis by the neighbor joining method.

  Specific examples of the method using the commercially available product include a method in which a commercially available live vaccine is inoculated into Vero cells and cultured. In this case, it is preferable to use a culture solution without trypsin.

Examples of PEDV belonging to Group I include P-5V strain (Sato T. et al., Virus Genes, 2011, Vol. 43 (1), pp. 72-78), CV777 strain (Pensaert MB). et al., Arch. Virol., 1978, Vol.58 (3), pp.243-247), DR13 strain (Park SJ et al., Virus Genes, 2007, Vol 35 (1), pp. 55-64), 96-P4C6 strain (Chemical and Serum Therapy Research Institute Swimgen registered trademark TGE / PED vaccine strain), JS-2004-2 strain (Zhao PD et al., Can. Res., 2015, Vol. 79 (1), 8-15), OH851 strain (Wang L. et. l., Emerg. Infect.Dis., 2014, Vol.20 (5), pp.917-919), CH / FJND-1 / 2011 strain (Tian Y., et al., Viruses, 2013, Vol.5). (8), pp. 1991-2004), DX strain (Zhao PD et al., Can. J. Vet. Res., 2015, Vol. 79 (1), 8-15) and the like.
Moreover, the said P-5V strain can also be easily culture | cultivated by the said method from Nisseiken PED live vaccine (made by Nisseiken).

  Examples of the PEDV belonging to the group II include MZ0116-2 / 2013 strain (see “Test Example 2” described later), OKN-1 / JPN / 2013 strain (Suzuki T. et al., Infect. Genet. Evol. 2015, Vol.36, pp.363-368), USA / Colorado / 2013 strain (Marthaler D. et.al., Genome Announce., 2013, Vol.1 (4), pp.e00555-13), USA / Minnesota 188/2014 strain (Marthaler D. et al., Emerg. Infect. Dis., 2014, Vol. 20 (12), pp. 2161-2163) and the like.

The method for attenuating the toxicity of PEDV is not particularly limited and may be appropriately selected from known methods according to the purpose. Examples thereof include a method of subculturing PEDV in a heterologous host. It is done.
The heterologous host is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cultured cells and embryonated chicken eggs.

The dose of the live vaccine of PEDV in the first administration step is not particularly limited, and the age, weight, constitution, symptom of the administration target individual, and whether or not a drug or drug containing other ingredients as active ingredients is administered. It can be appropriately selected in consideration of various factors, etc., but at a dose (10 ^7.0 TCID ₅₀ / dose or more) at which the virus content per administration becomes 10 ^7.0 TCID ₅₀ or more. Preferably there is. When the dose is less than 10 ^7.0 TCID ₅₀ / dose, PEDV-specific neutralizing antibody production and humoral immune response cannot be induced, and porcine epidemic diarrhea is prevented or treated. May not be possible.

<< Adjuvant >>
There is no restriction | limiting in particular as a kind of said adjuvant, According to the objective, it can select suitably, For example, aluminum salt, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, monophosphoryl lipid A (Monophosphoryl lipid A: MPL) , Microemulsion adjuvant, cholera toxin, E. coli heat-labile toxin, CpG oligonucleotide, polyinosine polycytidic acid (poly (I: C)), polymer adjuvant, squalene, dextrin derivative, liquid paraffin, tocopherol acetate, polysorbate, etc. It is done. The adjuvant may be used alone or in combination of two or more.
Among these, microemulsion adjuvant, cholera toxin, Escherichia coli heat-labile toxin, CpG oligonucleotide, polyinosine polycytidic acid (poly (I: C)), polymer adjuvant, squalene and the like are preferable, and microemulsion adjuvant is particularly preferable.

  The microemulsion adjuvant is composed of fine oily particles dispersed in water. The fine oily particles are particles obtained by mixing light mineral oil and a surfactant and emulsifying them in water to form particles of 5 nm to 500 nm.

  There is no restriction | limiting in particular as said light mineral oil, According to the objective, it can select suitably, For example, a liquid paraffin, an etherified liquid paraffin, a light liquid paraffin (Japanese Pharmacopoeia) etc. are mentioned. These light mineral oils may be used alone or in combination of two or more.

  Examples of the liquid paraffin include, under the trade name, MARCOL (trademark) 52 (trade name, manufactured by Exxon Mobil), Penreco (registered trademark) Drakeol (registered trademark) 6VR (trade name, manufactured by Penreco), and the like. .

  There is no restriction | limiting in particular as content of the said light mineral oil in the said microemulsion adjuvant, Although it can select suitably according to the objective, 0.001 mass%-10.0 mass% are preferable, 0.01 mass % To 0.1% by mass is more preferable. If the light mineral oil content is less than 0.001% by mass, the particles may become unstable. If it exceeds 10.0% by mass, the inoculum remains in the inoculated animal. May bring.

  The surfactant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, mannide monooleic acid, ethoxylated derivatives of oleic acid mannitan ester, glyceryl monostearate, decaglyceryl monolaurate Sorbitan monooleate, polyoxyethylene (20) sorbitan monopalmitate, and the like. These surfactants may be used alone or in combination of two or more.

  The content of the surfactant in the microemulsion adjuvant is not particularly limited as long as the light mineral oil can be emulsified into a microemulsion, and can be appropriately selected according to the purpose.

  The microemulsion adjuvant may further contain a chelating agent. When the microemulsion adjuvant contains a chelating agent, it is advantageous in that it has an effect of stabilizing the antigen (PEDV) bound to the fine oily fine particles and assisting the binding of the fine oily particles to immunocompetent cells. is there.

  The chelating agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include polyionic acid complexes such as calcium gluconate, manganese gluconate, aluminum salicylate, and soluble aluminum acetate. It is done. These chelating agents may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as content of the said chelating agent in the said microemulsion adjuvant, Although it can select suitably according to the objective, 0.002 mass%-30 mass% are preferable, 0.01 mass%- 15 mass% is more preferable. When the content of the chelating agent is less than 0.002% by mass, the immune response activity may not be enhanced, and when it exceeds 30% by mass, a side effect may be caused to the inoculated animal. is there.

  The method for preparing the microemulsion adjuvant containing the chelating agent is not particularly limited and can be appropriately selected according to the purpose. For example, the light mineral oil, the surfactant, and the water Examples include a method of adding a chelating agent and emulsifying by a known emulsifying means to form a microemulsion adjuvant. Specific examples include those described in JP-A Nos. 2000-131417 and 2005-75752. A method or the like can be used. Commercially available products may be used as the microemulsion adjuvant. Specific examples of commercially available products include MONTANIDE IMS series IMS 1312 VG, IMS 1313 VG, IMS 251C, IMS 2215 VG, IMS 3012 VG ST (all , Manufactured by SEPPIC).

  The dosage of the adjuvant in the first administration step is not particularly limited as long as the effect of the live vaccine of PEDV is not impaired, and the type of adjuvant and the administration of drugs and drugs containing other ingredients as active ingredients It can be appropriately selected in consideration of various factors such as presence / absence, age of an individual to be administered, weight, constitution, and symptoms.

  The dose when the adjuvant is the microemulsion adjuvant is not particularly limited, but the volume ratio of the microemulsion adjuvant to the virus solution containing the PEDV live vaccine is 20/80 to 80/20. A dose of 50/50 is particularly preferred. When the volume ratio (virus solution containing microemulsion adjuvant / PEDV live vaccine) is less than 20/80, production of PEDV-specific neutralizing antibodies and humoral immune response cannot be induced. In some cases, epidemic diarrhea cannot be prevented or treated, and if the ratio exceeds 80/20, swelling, induration, redness, fever, anaphylactic shock, etc. may occur at the administration site, and PEDV-specific neutralizing antibody production Or a humoral immune response cannot be induced, and porcine epidemic diarrhea cannot be prevented or treated.

  When the live vaccine of PEDV and the adjuvant are administered to pigs by either oral administration or nasal administration, the adjuvant effect of the adjuvant can be obtained and before the second administration step. The PEDV live vaccine and the adjuvant may be administered at the same time or may be administered separately, but it is convenient to administer at the same time. It is preferable at this point.

The method of administering the live vaccine of PEDV and the adjuvant simultaneously to the pig by either oral administration or nasal administration is not particularly limited and can be appropriately selected according to the purpose. Examples include a method of administering a composition obtained by mixing a live vaccine and the adjuvant.
As the composition in which the live PEDV vaccine and the adjuvant are mixed, the vaccine for oral or nasal administration of the present invention described later is preferably used.

When the live vaccine of PEDV and the adjuvant are separately administered to pigs by either oral administration or nasal administration, the adjuvant effect of the adjuvant is obtained as the administration order of the live vaccine of PEDV and the adjuvant. As long as it is possible, it is not particularly limited and can be appropriately selected depending on the purpose.
Further, the administration interval between the live vaccine of PEDV and the adjuvant is not particularly limited as long as the adjuvant effect of the adjuvant is obtained, and can be appropriately selected according to the purpose.

  There is no restriction | limiting in particular as the frequency | count of performing the said 1st administration process, According to the objective, it can select suitably. The present invention is advantageous in that a sufficient effect can be obtained only by performing the first administration step once.

<Second administration step>
The intramuscular administration step is a step of administering the inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant to the pig after the first administration step by intramuscular administration.

<< Inactivated vaccine of swine epidemic diarrhea virus (PEDV) >>
An inactivated vaccine of PEDV is a vaccine in which the pathogenicity of PEDV is eliminated or the toxin is detoxified.
There is no restriction | limiting in particular as a classification | category of PEDV used for the said inactivated vaccine of PEDV, a obtaining method, etc., It can select suitably according to the objective, For example, the live vaccine of PEDV in the said 1st administration process And the like described in the item.

The method for inactivating the infectivity of PEDV is not particularly limited and may be appropriately selected from known methods according to the purpose. For example, a method of treating PEDV with a drug such as formalin, heating And a method of changing pH, a method of irradiating gamma rays, a method of irradiating ultraviolet rays, and the like.
The PEDV used for the inactivated vaccine of PEDV may inactivate the isolated virus as it is, or may inactivate the attenuated PEDV.

The dose of the inactivated vaccine of PEDV in the second administration step is not particularly limited, and is the administration of a drug or drug containing as an active ingredient the age, weight, constitution, symptom, and other ingredients of the individual to be administered. The dose can be appropriately selected in consideration of various factors such as presence or absence, but the dose per virus dose is 10 ^6.5 TCID ₅₀ or more (10 ^6.5 TCID ₅₀ / dose or more) A dose of 10 ^7.5 TCID ₅₀ or more (10 ^7.5 TCID ₅₀ / dose or more) is more preferable. The dose is less than 10 6.5 ^TCID 50 _/ dose, it can not induce the production of PEDV specific neutralizing antibodies and humoral immune response, preventing or treating porcine epidemic diarrhea May not be possible.

<< Adjuvant >>
There is no restriction | limiting in particular as a kind of said adjuvant, According to the objective, it can select suitably, For example, what was described in the item of the adjuvant in the said 1st administration process etc. are mentioned.
Among these, aluminum salts, microemulsion adjuvants, polymer adjuvants, dextrin derivatives, liquid paraffin, squalene, tocopherol acetate, polysorbate and the like are preferable, and microemulsion adjuvants are particularly preferable.

  The mode of the microemulsion adjuvant is the same as that described in the adjuvant item in the first administration step.

  The dosage of the adjuvant in the second administration step is not particularly limited as long as it does not impair the effect of the inactivated vaccine of PEDV, and the type of adjuvant, administration of a drug or drug containing other ingredients as active ingredients It can be appropriately selected in consideration of various factors such as presence / absence, age of the subject individual, body weight, constitution, and symptoms.

  The dose when the adjuvant is the microemulsion adjuvant is not particularly limited, but the volume ratio of the microemulsion adjuvant to the virus solution containing the inactivated vaccine of PEDV is 20/80 to 80 / A dose of 20 is preferred, and a dose of 50/50 is particularly preferred. When the volume ratio (microemulsion adjuvant / virus solution containing an inactivated vaccine of PEDV) is less than 20/80, PEDV-specific neutralizing antibody production and humoral immune response cannot be induced, It may not be possible to prevent or treat swine epidemic diarrhea, and if it exceeds 80/20, swelling, induration, redness, fever, anaphylactic shock, etc. may occur at the administration site, and PEDV-specific neutralizing antibody Production and humoral immune responses cannot be induced, and porcine epidemic diarrhea cannot be prevented or treated.

  There is no particular limitation on the time when the inactivated vaccine of PEDV and the adjuvant are administered to the pig by intramuscular administration as long as the adjuvant effect of the adjuvant is obtained and after the first administration step. The PEDV inactivated vaccine and the adjuvant may be administered at the same time or separately, but it is convenient to administer at the same time. preferable.

The method of administering the inactivated vaccine of PEDV and the adjuvant simultaneously to the pig by intramuscular administration is not particularly limited and can be appropriately selected depending on the purpose. For example, the inactivated vaccine of PEDV and And a method of administering a composition mixed with the adjuvant.
As the composition in which the inactivated vaccine of PEDV and the adjuvant are mixed, the intramuscular vaccine of the present invention described later is suitably used.

When the inactivated vaccine of PEDV and the adjuvant are separately administered to the pig by intramuscular administration, the order of administration of the inactivated vaccine of PEDV and the adjuvant is as long as the adjuvant effect of the adjuvant is obtained. There is no restriction | limiting in particular, According to the objective, it can select suitably.
Moreover, the administration interval between the inactivated vaccine of PEDV and the adjuvant is not particularly limited as long as the adjuvant effect of the adjuvant is obtained, and can be appropriately selected according to the purpose.

  There is no restriction | limiting in particular as the frequency | count of performing the said 2nd administration process, According to the objective, it can select suitably. The present invention is advantageous in that a sufficient effect can be obtained only by performing the second administration step once.

  The administration interval between the first administration step and the second administration step is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 week to 20 weeks, and is preferably 2 weeks to 10 weeks. Weeks are more preferred, and 4 to 10 weeks are particularly preferred. When the administration interval is less than one week, production of PEDV-specific neutralizing antibodies and humoral immune response cannot be induced, and porcine epidemic diarrhea may not be prevented or treated, Even if it exceeds 20 weeks, production of PEDV-specific neutralizing antibodies and humoral immune response cannot be induced, and pig epidemic diarrhea may not be prevented or treated.

(Vaccine kit)
The vaccine kit of the present invention comprises a live vaccine of porcine epidemic diarrhea virus for oral or nasal administration, an adjuvant for oral or nasal administration, an inactivated vaccine of porcine epidemic diarrhea virus for intramuscular administration, and intramuscular It has an adjuvant for administration, and it has other configurations as required.

<Live porcine epidemic diarrhea virus (PEDV) vaccine for oral or nasal administration>
There is no particular limitation on the classification, acquisition method, and method for attenuating the toxicity of PEDV used in the PEDV live vaccine for oral or nasal administration, and it can be appropriately selected according to the purpose. Examples thereof include those described in the item of live vaccine for PEDV in the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above.

There is no restriction | limiting in particular as virus content of the said PEDV live vaccine for oral or nasal administration in the said vaccine kit, According to the frequency | count of use etc., it can select suitably. The virus content per administration of the PEDV live vaccine for oral or nasal administration is preferably 10 ^7.0 TCID ₅₀ / dose or more. In addition, the PEDV live vaccine for oral or nasal administration is the age, weight, constitution, symptom of the administration subject, whether or not a drug or drug is administered as an active ingredient at the time of using the vaccine kit, etc. In consideration of various factors, it can be appropriately adjusted by selective dilution or the like.

<Adjuvant for oral or nasal administration>
The type of adjuvant for oral or nasal administration is not particularly limited and can be appropriately selected depending on the purpose. For example, the first administration of the above-described method for preventing or treating swine epidemic diarrhea of the present invention What was described by the adjuvant item in the process is mentioned.
Among these, microemulsion adjuvant, cholera toxin, Escherichia coli heat-labile toxin, CpG oligonucleotide, polyinosine polycytidic acid (poly (I: C)), polymer adjuvant, squalene and the like are preferable, and microemulsion adjuvant is particularly preferable.

  The mode of the microemulsion adjuvant is the same as that described in the adjuvant item in the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above.

  The amount of the adjuvant for oral or nasal administration in the vaccine kit is not particularly limited, and is appropriately selected according to the number of times of use, the amount of virus solution containing the PEDV live vaccine for oral or nasal administration, and the like. be able to.

The vaccine kit may have the PEDV live vaccine for oral or nasal administration and the adjuvant for oral or nasal administration as separate components, and in the state of a composition in which these are mixed. You may have.
As the composition in which the live oral PEDV vaccine for oral or nasal administration and the adjuvant for oral or nasal administration are mixed, the oral or nasal administration vaccine of the present invention described later is preferably used.

<Inactivated vaccine of swine epidemic diarrhea virus (PEDV) for intramuscular administration>
There is no particular limitation on the classification and acquisition method of PEDV used in the PEDV inactivated vaccine for intramuscular administration, the method of inactivating the infectivity of PEDV, and it can be appropriately selected according to the purpose. For example, the aspect described in the item of the inactivated vaccine of PEDV in the 2nd administration process of the prevention or treatment method of the above-mentioned pig epidemic diarrhea of this invention is mentioned.

There is no restriction | limiting in particular as virus content of the said PEDV inactivation vaccine for intramuscular administration in the said vaccine kit, According to the frequency | count of use etc., it can select suitably. Viral content per dose of one PEDV inactivated vaccine for administration the ^{intramuscular,} preferably 10 6.5 TCID ₅₀ / dose or ^more, more preferably 10 7.5 TCID ₅₀ / dose or more. In addition, the PEDV inactivated vaccine for intramuscular administration, when using the vaccine kit, the age, weight, constitution, symptom of the administration subject individual, the presence or absence of administration of a medicine or drug containing other ingredients, In consideration of various factors, it can be appropriately adjusted by selective dilution or the like.

<Adjuvant for intramuscular administration>
The type of the intramuscular adjuvant is not particularly limited and can be appropriately selected depending on the purpose. For example, the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above. And the like described in the adjuvant item.
Among these, aluminum salts, microemulsion adjuvants, polymer adjuvants, dextrin derivatives, liquid paraffin, squalene, tocopherol acetate, polysorbate and the like are preferable, and microemulsion adjuvants are particularly preferable.

  The mode of the microemulsion adjuvant is the same as that described in the adjuvant item in the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above.

  There is no restriction | limiting in particular as the quantity of the adjuvant for intramuscular administration in the said vaccine kit, It can select suitably according to the frequency | count of use, the quantity of the virus liquid containing the PEDV inactivation vaccine for the said intramuscular administration, etc.

The vaccine kit may have an inactivated PEDV vaccine for intramuscular administration and an adjuvant for intramuscular administration as separate components, and has a composition in which these are mixed. May be.
As the composition in which the inactivated PEDV vaccine for intramuscular administration and the adjuvant for intramuscular administration are mixed, the intramuscular vaccine of the present invention described later is suitably used.

<Other configurations>
The other configuration is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, a solution, a physiological saline for dilution, a container for preparing an event, a syringe , Injection needles, package inserts, etc.

  There is no restriction | limiting in particular as a composition of the said solution, It can select suitably according to the objective, For example, what is described in the item of the other component in the vaccine for oral or nasal administration of this invention mentioned later Etc.

<How to use>
The vaccine kit comprises an oral or nasal administration of the PEDV live vaccine for oral or nasal administration and the oral or nasal adjuvant, and then the inactivated PEDV vaccine for intramuscular administration and the An intramuscular adjuvant is used by intramuscular administration.

  The oral vaccine for oral or nasal administration of PEDV and the adjuvant for oral or nasal administration may be administered simultaneously or separately, but it is convenient to administer simultaneously. preferable.

  When the oral vaccine for oral or nasal administration of PEDV and the adjuvant for oral or nasal administration are simultaneously administered to pigs by either oral administration or nasal administration, PEDV for oral or nasal administration is used. It is preferable to use the composition obtained by mixing the live vaccine and the adjuvant for oral or nasal administration.

When the oral vaccine for oral or nasal administration of PEDV and the adjuvant for oral or nasal administration are separately administered to pigs by either oral administration or nasal administration, PEDV for oral or nasal administration is used. The order of administration of the live vaccine and the adjuvant for oral or nasal administration is not particularly limited and may be appropriately selected depending on the purpose.
The interval between the oral vaccine for oral or nasal administration of PEDV and the adjuvant for oral or nasal administration is not particularly limited as long as the adjuvant effect of the adjuvant for oral or nasal administration is obtained. Can be appropriately selected according to the purpose.

  The inactivated PEDV vaccine for intramuscular administration and the adjuvant for intramuscular administration may be administered at the same time or separately, but it is preferable in that it is convenient to administer at the same time.

  When the inactivated PEDV vaccine for intramuscular administration and the adjuvant for intramuscular administration are simultaneously administered to the pig by intramuscular administration, the inactivated vaccine of PEDV for intramuscular administration and the intramuscular administration It is preferable to use the composition mixed with an adjuvant.

When the inactivated PEDV vaccine for intramuscular administration and the adjuvant for intramuscular administration are separately administered to the pig by intramuscular administration, the inactivated vaccine of PEDV for intramuscular administration and the intramuscular administration There is no restriction | limiting in particular as an administration order with an adjuvant, According to the objective, it can select suitably.
In addition, the administration interval between the inactivated PEDV vaccine for intramuscular administration and the adjuvant for intramuscular administration is not particularly limited as long as the adjuvant effect of the intramuscular administration adjuvant is obtained. It can be selected appropriately.

  The vaccine kit can be suitably used for the method for preventing or treating swine epidemic diarrhea of the present invention described above.

(Vaccine for oral or nasal administration)
The vaccine for oral or nasal administration of the present invention contains a live vaccine of porcine epidemic diarrhea virus and an adjuvant, and further contains other components as necessary.
The vaccine for oral or nasal administration is used for pigs given booster immunization after intramuscular administration of an inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant.

<Live vaccine of porcine epidemic diarrhea virus (PEDV)>
There are no particular restrictions on the classification, acquisition method, and method for attenuating the toxicity of PEDV used in live PEDV vaccines, and can be appropriately selected according to the purpose. The aspect described in the item of the live vaccine of PEDV in the 1st administration process of the prevention or treatment method of swine epidemic diarrhea, etc. are mentioned.

The content of the live PEDV vaccine in the oral or nasal vaccine is not particularly limited and may be appropriately selected depending on the intended purpose, but is 10 ^7.0 TCID ₅₀ / dose or more. preferable. When the content is less than 10 ^7.0 TCID ₅₀ / dose, PEDV-specific neutralizing antibody production and humoral immune response cannot be induced, and porcine epidemic diarrhea is prevented or treated. May not be possible.

<Adjuvant>
The type of the adjuvant is not particularly limited and may be appropriately selected depending on the purpose. For example, the adjuvant item in the first administration step of the above-described method for preventing or treating swine epidemic diarrhea of the present invention is as follows. And the like described above.
Among these, microemulsion adjuvant, cholera toxin, Escherichia coli heat-labile toxin, CpG oligonucleotide, polyinosine polycytidic acid (poly (I: C)), polymer adjuvant, squalene and the like are preferable, and microemulsion adjuvant is particularly preferable.

  The mode of the microemulsion adjuvant is the same as that described in the adjuvant item in the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above.

  The content of the adjuvant in the oral or nasal vaccine is not particularly limited as long as the adjuvant effect is obtained without impairing the effect of the live PEDV vaccine, and can be appropriately selected according to the purpose. .

  When the adjuvant is the microemulsion adjuvant, the content of the microemulsion adjuvant in the vaccine for oral or nasal administration is not particularly limited and can be appropriately selected according to the purpose. % To 80% by volume is preferable, and 50% by volume is particularly preferable. When the content of the microemulsion is less than 20% by volume, the production of PEDV-specific neutralizing antibodies and the humoral immune response cannot be induced, and porcine epidemic diarrhea cannot be prevented or treated. If it exceeds 80% by volume, it may cause swelling, induration, redness, fever, anaphylactic shock, etc. at the administration site, production of neutralizing antibodies specific to PEDV, and humoral immune response. In other cases, it is impossible to prevent or treat swine epidemic diarrhea.

<Other ingredients>
There is no restriction | limiting in particular as said other component, According to the objective, it can select suitably from the carriers accept | permitted pharmacologically, For example, an additive, an adjuvant, water, etc. are mentioned. These may be used alone or in combination of two or more.

  The additive or the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bactericides, preservatives, binders, thickeners, fixing agents, binders, and coloring agents. , Stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, preservatives, and the like.

  There is no restriction | limiting in particular as said disinfectant, According to the objective, it can select suitably, For example, cationic surfactants, such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, etc. are mentioned.

  There is no restriction | limiting in particular as said preservative, According to the objective, it can select suitably, For example, paraoxybenzoic acid esters, chlorobutanol, cresol, thimerosal, phenoxyethanol etc. are mentioned.

  The binder, the thickener, or the sticking agent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose , Hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol ester alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, Ethylene / propylene block polymer, sodium polyacrylate, polyvinylpyrrolidone, etc. And the like.

  The binder is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxy Examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.

  The colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.

  The stabilizer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tragacanth, gum arabic, gelatin, sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Is mentioned.

  There is no restriction | limiting in particular as said pH adjuster or said buffering agent, According to the objective, it can select suitably, For example, sodium citrate, sodium acetate, sodium phosphate etc. are mentioned.

  There is no restriction | limiting in particular as said tonicity agent, According to the objective, it can select suitably, For example, sodium chloride, potassium chloride, glucose etc. are mentioned.

  There is no restriction | limiting in particular as content of the said other component in the said vaccine for oral or nasal administration, unless the effect of this invention is impaired, It can select suitably according to the objective.

<Dosage form>
The dosage form of the vaccine for oral or nasal administration is not particularly limited as long as it can be administered orally or nasally, and can be appropriately selected according to the purpose. Examples thereof include solid preparations and liquid preparations. It is done. Among these, as said vaccine for oral administration, the said solid agent, the said liquid agent, etc. are preferable, and as said vaccine for nasal administration, the said liquid agent is preferable.

-Solid agent-
There is no restriction | limiting in particular as said solid agent, According to the objective, it can select suitably, For example, a hard capsule, a soft capsule, etc. are mentioned.

-Liquid-
The liquid preparation is not particularly limited and may be appropriately selected depending on the intended purpose. However, when used as a vaccine for oral administration, for example, a syrup, a drink, a suspension, an alcoholic beverage, a feed preparation, etc. Can be mentioned.
When the liquid is used as a vaccine for nasal administration, examples thereof include liquids, eye drops, aerosols, and sprays.

<Manufacturing method>
The method for producing the vaccine for oral or nasal administration is not particularly limited as long as the live vaccine of PEDV and the adjuvant can be mixed. It can be selected as appropriate according to the conditions.

<Use>
The oral or nasal vaccine is used for pigs given booster immunization after intramuscular administration of an inactivated PEDV vaccine and an adjuvant.
The method for giving booster immunity to the pig is not particularly limited and may be appropriately selected depending on the intended purpose. However, the method described in the second step of the method for preventing or treating swine epidemic diarrhea is not limited. Preferably used.

  The oral or nasal vaccine may be used singly or in combination with a medicament containing other ingredients as active ingredients. In addition, the oral or nasal vaccine may be used in a state where it is blended in a medicine, feed, drinking water or the like containing other ingredients as active ingredients.

  The vaccine for oral or nasal administration can induce the production of neutralizing antibodies specific for PEDV by giving booster immunization by intramuscular administration of an inactivated vaccine of PEDV and an adjuvant. Furthermore, production of IgA specific to PEDV can also be induced by a humoral immune response, so that porcine epidemic diarrhea can be efficiently prevented or treated.

  The oral or nasal vaccine can be administered not only to mother pigs but also to piglets, so it can be used for piglets that cannot take milk for some reason, or against PEDV in the milk of mother pigs. Even when the concentration of the neutralizing antibody is low, it is advantageous in that the infection of PEDV in piglets can be prevented or treated.

(Intramuscular vaccine)
The vaccine for intramuscular administration of the present invention contains an inactivated vaccine for swine epidemic diarrhea virus and an adjuvant, and further contains other components as necessary.
The intramuscular vaccine is used for pigs given the first immunization by oral or nasal administration of live porcine epidemic diarrhea virus vaccine and adjuvant.

<Inactivated vaccine of porcine epidemic diarrhea virus (PEDV)>
There are no particular restrictions on the classification, acquisition method, and method for inactivating the PEDV infectivity of PEDV used in inactivated PEDV vaccines, which can be appropriately selected according to the purpose. Examples include the aspect described in the item of inactivated vaccine for PEDV in the second administration step of the method for preventing or treating swine epidemic diarrhea of the invention.

The content of the inactivated vaccine of the PEDV in the intramuscular vaccine is not particularly limited, suitably it can be ^selected, preferably 10 6.5 TCID ₅₀ / dose or more depending on the purpose, ^{107 .5} TCID ₅₀ / dose or more is more preferable. When the content is less than 10 ^6.5 TCID ₅₀ / dose, production of PEDV-specific neutralizing antibodies and humoral immune response cannot be induced, and porcine epidemic diarrhea can be prevented or treated. There are things that cannot be done.

<Adjuvant>
The type of the adjuvant is not particularly limited and may be appropriately selected depending on the purpose. For example, the adjuvant item in the first administration step of the above-described method for preventing or treating swine epidemic diarrhea of the present invention is as follows. And the like described above.
Among these, aluminum salts, microemulsion adjuvants, polymer adjuvants, dextrin derivatives, liquid paraffin, squalene, tocopherol acetate, polysorbate and the like are preferable, and microemulsion adjuvants are particularly preferable.

  The mode of the microemulsion adjuvant is the same as that described in the adjuvant item in the first administration step of the method for preventing or treating swine epidemic diarrhea of the present invention described above.

  The content of the adjuvant in the intramuscular vaccine is not particularly limited as long as the adjuvant effect is obtained without impairing the effect of the inactivated vaccine of PEDV, and can be appropriately selected according to the purpose.

  When the adjuvant is the microemulsion adjuvant, the content of the microemulsion adjuvant in the intramuscular vaccine is not particularly limited and may be appropriately selected depending on the intended purpose. 80% by volume is preferable, and 50% by volume is particularly preferable. When the content of the microemulsion is less than 20% by volume, the production of PEDV-specific neutralizing antibodies and the humoral immune response cannot be induced, and porcine epidemic diarrhea cannot be prevented or treated. If it exceeds 80% by volume, it may cause swelling, induration, redness, fever, anaphylactic shock, etc. at the administration site, production of neutralizing antibodies specific to PEDV, and humoral immune response. In other cases, it is impossible to prevent or treat swine epidemic diarrhea.

<Other ingredients>
The other components are not particularly limited and can be appropriately selected from pharmacologically acceptable carriers according to the purpose. For example, other components of the oral or nasal vaccine And the like described in.

  There is no restriction | limiting in particular as content of the said other component in the said vaccine for intramuscular administration, unless the effect of this invention is impaired, It can select suitably according to the objective.

<Dosage form>
The dosage form of the intramuscular vaccine is not particularly limited as long as it can be administered intramuscularly, and can be appropriately selected according to the purpose. Examples thereof include injections.

-Injection-
There is no restriction | limiting in particular as said injection, According to the objective, it can select suitably, For example, a solution, a suspension, the solid agent for use, etc. are mentioned.

<Manufacturing method>
The method for producing the intramuscular vaccine is not particularly limited as long as the inactivated vaccine of PEDV and the adjuvant can be mixed, and can be selected from known methods according to the dosage form. It can be appropriately selected depending on the case.

<Use>
The intramuscular vaccine is used for pigs given primary immunization by oral or nasal administration with live PEDV vaccine and adjuvant.
The method for conferring initial immunity to the pig is not particularly limited and may be appropriately selected depending on the purpose. However, the method described in the first step of the method for preventing or treating swine epidemic diarrhea is described above. Preferably used.

  The said intramuscular vaccine may be used individually by 1 type, and may be used together with the pharmaceutical which uses another component as an active ingredient. Moreover, the vaccine for intramuscular administration may be used in a state of being blended in a medicine containing other ingredients as active ingredients.

  The intramuscular vaccine is used for pigs given the initial immunization by oral or nasal administration of a live PEDV vaccine and an adjuvant, thereby producing neutralizing antibodies specific for PEDV. In addition, the production of IgA specific to PEDV can also be induced by a humoral immune response, so that porcine epidemic diarrhea can be efficiently prevented or treated.

  Since the intramuscular vaccine can be directly administered not only to mother pigs but also to piglets, neutralization against PEDV in piglets that cannot take milk for some reason or mother pigs' milk Even when the antibody concentration is low, it is advantageous in that the infection of PEDV in piglets can be prevented or treated.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Production Example 1)
<Production of PEDV (P-5V strain) live vaccine 1>
<< Separation and Production of PEDV P-5V Strain >>
The small intestine was collected from a piglet that developed porcine epidemic diarrhea (PED) in the field to obtain a 10 mass% tissue emulsion. This 10% tissue emulsion was orally administered to piglets not infected with PED, and after 2 days, the small intestine was collected to prepare a 10 mass% tissue emulsion. In the same manner, the piglets not infected with PED were passaged up to 3 generations. Trypsin is added to the tissue emulsion obtained from the third generation piglet so that the final concentration is 10 μg / mL, and inoculated into Vero cells previously confluent in a 25 cm ² culture flask and incubated at 37 ° C. for 60 minutes. Cultured. Thereafter, 5 mL of a culture solution prepared by the method described below (hereinafter sometimes referred to as “10 μg / mL trypsin-added culture solution”) was added, and the mixture was cultured at 37 ° C. The cytopathic effect (CPE) was confirmed daily with an optical microscope, and when CPE was confirmed, the culture was stopped and passaged. In passage of the isolated virus, the amount of trypsin added was gradually decreased as the passage proceeded, and finally, trypsin was not added, and passage was performed up to a total passage number of 100.
-10 μg / mL trypsin culture solution-
Yeast extract (Difco Laboratories) and 0.03% (w / v) in 500 mL of Minimal Essential Media (MEM) (Thermo Fisher Scientific) to a final concentration of 0.02% (w / v). The tryptic phosphate broth (manufactured by Difco Laboratories) was dissolved so that the concentration of glycine was reduced to 50,000, penicillin 50,000 units, streptomycin 50 mg, and trypsin to a final concentration of 10 μg / mL.

<< Identification of P-5V strain >>
Viral RNA was extracted from the culture supernatant of the P-5V strain using a TRIZol (registered trademark) reagent (manufactured by Thermo Fisher Scientific). RT using PEDV RNA using SuperScript (registered trademark) III One-Step RT-PCR System (manufactured by Thermo Fisher Scientific) and eight PEDV S gene-specific primer sets (primer sets 1 to 8) shown in Table 1 below -Amplification was performed by PCR with a thermal cycler (Veriti 96-Well Thermal Cycler, manufactured by Thermo Fisher Scientific). The amplified RT-PCR product was transformed into pCR (registered trademark) 2.1-TOPO TA vector (Thermo Fisher Scientific) using E. coli TOP10 (Thermo Fisher Scientific) and cloned. . Escherichia coli was cultured overnight at 37 ° C. using LB medium (composition: sodium chloride 10 g, Bacto Trypton 10 g, yeast extract 5 g dissolved in 1 L distilled water), and then QIAprep Spin Miniprep Kit (manufactured by QIAGEN) was used. Used to purify the plasmid. The PEDV S gene base sequence was decoded from the purified plasmid using BigDye (registered trademark) Terminator v3.1 reagent and 3130xl genetic analyzer (Applied Biosystems). Each decoded sequence fragment was assembled, and phylogenetic tree analysis was performed by the neighbor joining method using MEGA 4.0 software (http://www.megasoftware.net).
As a result, the strain obtained above was identified as PEDV genetic group I P-5V strain (see FIG. 1).

<< Preparation of PEDV (P-5V strain) concentrated virus antigen solution 1 >>
The P-5V strain was inoculated into Vero cells and cultured at 37 ° C. for 3 days. The Vero cells were recovered and freeze-thawed. Subsequently, the freeze-thawed product was centrifuged, and the resulting supernatant was recovered as “PEDV (P-5V strain) virus antigen solution 1”. 7.5% (w / v) polyethylene glycol 6000 and 1M sodium chloride were added to the PEDV (P-5V strain) virus antigen solution 1, and the mixture was stirred overnight at 4 ° C., and then centrifuged. Separated and recovered the precipitate. The precipitate was dissolved in 1/20 volume (v / v) physiological saline of PEDV (P-5V strain) virus antigen solution 1, and then centrifuged again. The resulting supernatant was treated with “PEDV (P- 5V strain) concentrated virus antigen solution 1 ”.

-Measurement of virus titer-
The virus titer (virus infectivity titer) of the PEDV (P-5V strain) concentrated virus antigen solution 1 was measured by the 50% tissue culture infectious dose (TCID ₅₀ ) method.
Specifically, Vero cells were seeded on a 24-well cell culture plate at 0.75 × 10 ⁶ cells / 0.5 ml / well and cultured at 37 ° C. for 3 days. The antigen solution to be measured (here, the PEDV (P-5V strain) concentrated virus antigen solution 1) was added to a culture solution (500 mL of MEM (Thermo Fisher Scientific)) at a final concentration of 0.02% (w / v). The yeast extract (Difco Laboratories) and tryptophosphate phosphate broth (Difco Laboratories) were dissolved so that the final concentration was 0.03% (w / v), and then penicillin 50,000 units and streptomycin 50 mg. Was diluted 10-fold in a step, and inoculated into Vero cells, followed by incubation at 37 ° C. for 90 minutes. After exchanging the culture solution, the cells were cultured at 37 ° C. for 7 days. The cytopathic effect (CPE) was observed with an optical microscope, and the virus titer (TCID ₅₀ ) was calculated according to the Reed-Muench method.
As a result, the virus titer of the PEDV (P-5V strain) concentrated virus antigen solution 1 was 10 ^7.75 TCID ₅₀ / mL.

<< Preparation of PEDV (P-5V strain) live vaccine 1 >>
The PEDV (P-5V strain) concentrated virus antigen solution 1 and a microemulsion (trade name: MONTANIDE IMS1313 VG, manufactured by SEPPIC) were mixed in an equal amount (v / v) at room temperature (25 ± 5 ° C.). It stirred for 5 minutes or more and was set as "PEDV (P-5V strain) live vaccine 1".

(Production Example 2)
<Production of PEDV (P-5V strain) live vaccine 2>
<< Preparation of PEDV (P-5V strain) concentrated virus antigen solution 2 >>
The PEDV strain P-5V isolated and produced in Production Example 1 was inoculated into Vero cells. After culturing at 37 ° C. for 3 days, Vero cells were recovered and freeze-thawed. Next, the frozen and thawed product was centrifuged, and the resulting supernatant was recovered as “PEDV (P-5V strain) virus antigen solution 2”. 7.5% (w / v) polyethylene glycol 6000 and 1M sodium chloride were added to the PEDV (P-5V strain) virus antigen solution 2 respectively, and the mixture was stirred overnight at 4 ° C., and then centrifuged. Separated and recovered the precipitate. The precipitate was dissolved in 1/20 volume (v / v) physiological saline of the PEDV (P-5V strain) virus antigen solution 2, and then centrifuged again. The resulting supernatant was treated with PEDV (P- 5V strain) concentrated virus antigen solution 2 ”.

The virus titer of the PEDV (P-5V strain) concentrated virus antigen solution 2 was measured by the TCID ₅₀ method described in the column “Measurement of virus titer” in Production Example 1. As a result, the virus titer of the PEDV (P-5V strain) concentrated virus antigen solution 2 was 10 ^7.75 TCID ₅₀ / mL.

<< Preparation of PEDV (P-5V strain) live vaccine 2 >>
The PEDV (P-5V strain) concentrated virus antigen solution 2 and the microemulsion prepared in Production Example 1 are mixed in an equal amount (v / v) and stirred at room temperature (25 ± 5 ° C.) for 5 minutes or more. "PEDV (P-5V strain) live vaccine 2".

(Production Example 3)
<Production of PEDV (P-5V strain) inactivated vaccine 1>
<< Preparation of PEDV (P-5V strain) inactivated virus antigen solution 1 >>
Neutral buffered formalin (manufactured by Kanto Chemical Co., Inc.) was applied to PEDV (P-5V strain) concentrated virus antigen solution 1 obtained in Production Example 1 so that the final concentration was 0.2% (v / v). The PEDV (P-5V strain) virus antigen was inactivated by adding and stirring at 4 ° C. for 48 hours to obtain “PEDV (P-5V strain) inactivated virus antigen solution 1”.

<< Preparation of PEDV (P-5V strain) inactivated vaccine 1 >>
The PEDV (P-5V strain) inactivated virus antigen solution 1 and the microemulsion prepared in Production Example 1 are mixed in an equal amount (v / v) and stirred at room temperature (25 ± 5 ° C.) for 5 minutes or more. Thus, it was referred to as “PEDV (P-5V strain) inactivated vaccine 1”.

(Production Example 4)
<Production of PEDV (P-5V strain) inactivated vaccine 2>
<< Preparation of PEDV (P-5V strain) inactivated virus antigen solution 2 >>
Neutral buffered formalin (manufactured by Kanto Chemical Co., Ltd.) is used so that the final concentration is 0.2% (v / v) with respect to the PEDV (P-5V strain) concentrated virus antigen solution 2 obtained in Production Example 2. The PEDV (P-5V strain) virus antigen was inactivated by adding and stirring at 4 ° C. for 48 hours to obtain “PEDV (P-5V strain) inactivated virus antigen solution 2”.

<< Preparation of PEDV (P-5V strain) inactivated vaccine 2 >>
The PEDV (P-5V strain) inactivated virus antigen solution 2 and the microemulsion prepared in Production Example 1 were mixed in equal amounts (v / v) and stirred at room temperature (25 ± 5 ° C.) for 5 minutes or more. Thus, it was named “PEDV (P-5V strain) inactivated vaccine 2”.

Example 1
<First immunization>
8-week-old pigs PED antibody negative, PEDV prepared in Preparation Example 1 (P-5V Ltd.) live vaccine 1 ^{(10mL, 10} 8.0 TCID ₅₀ / dose) were administered once orally.

<Booster>
Four weeks after the initial immunization, the PEDV (P-5V strain) inactivated vaccine 1 (2 mL, 10 ^7.5 TCID ₅₀ / dose) produced in Production Example 3 was intramuscularly administered (injected) once.

(Comparative Example 1)
<First immunization>
8-week-old pigs PED antibody negative were administered orally once a PEDV obtained in Production Example 1 (P-5V Ltd.) concentrate viral antigen solution ^{(10mL, 10} 8.0 TCID ₅₀ / dose).

<Booster>
Four weeks after the initial immunization, the PEDV (P-5V strain) inactivated vaccine 1 (2 mL, 10 ^7.5 TCID ₅₀ / dose) produced in Production Example 3 was intramuscularly administered (injected) once.

(Comparative Example 2)
<First immunization>
A Nisseiken PED live vaccine (Nisseiken Co., Ltd.) (2 mL) was intramuscularly administered (injected) once to a PED antibody-negative 8-week-old pig.

<Booster>
Four weeks after the initial immunization, the Nisseiken PED live vaccine (manufactured by Nisseiken) (2 mL) was intramuscularly administered (injected) once.

(Test Example 1)
For the pigs of Example 1, Comparative Example 1, and Comparative Example 2, blood was collected immediately before the first immunization and one week after the booster immunization. As controls, blood was collected at 8 weeks, 12 weeks, and 13 weeks of age for PED antibody-negative 8-week-old pigs that were not subjected to primary immunization or booster immunization. Next, the measurement of the PEDV neutralizing antibody titer in the collected blood and the induction of IgA production against PEDV were examined by the method described below.

<Measurement of PEDV neutralizing antibody titer>
Vero cells were seeded on a 24-well cell culture plate at 0.75 × 10 ⁶ cells / 0.5 ml / well and cultured at 37 ° C. for 3 days. A yeast extract (manufactured by Difco Laboratories) and a final concentration of 0.03% (w / v) in a culture solution (500 mL of MEM (manufactured by Thermo Fisher Scientific) to a final concentration of 0.02% (w / v)) Equivalent amount of P-5V strain (200 TCID) in swine serum diluted 2-fold stepwise with tryptophosphate phosphate broth (Difco Laboratories) and penicillin (50,000 units, streptomycin 50 mg). ₅₀ ) was mixed and reacted at 37 ° C. for 90 minutes. A mixed solution of serum and P-5V strain was inoculated into the Vero cells and cultured at 37 ° C. for 90 minutes. After exchanging the culture solution, the cells were cultured at 37 ° C. for 7 days. The cytopathic effect (CPE) was observed with an optical microscope to measure the PEDV neutralizing antibody titer. The results are shown in Table 3 below. The unit of neutralizing antibody titer is indicated by the dilution ratio (times) of serum.

<Induction of IgA production against PEDV>
<< Detection of IgA against PEDV by Western blotting >>
The blood collected one week after the booster in Example 1, Comparative Example 1 and Comparative Example 2 was centrifuged to separate the serum. 200 μL of a virus solution containing the P-5V strain (10 ^7.0 TCID ₅₀ / mL) was run on an SDS-PAGE gel and then blotted onto a poly (vinylidene fluoride) (PVDF) filter (Merck Millipore). PBS to which 3% (w / v) skim milk and 0.05% (w / v) Tween 20 were added as primary antibodies (composition: NaCl 8 g, KCl 0.2 g, Na ₂ HPO ₄ / 12H ₂ O 2.9 g) , And 0.2 g of KH ₂ PO ₄ dissolved in 1 L of distilled water (with the same composition in the following test examples), swine serum diluted 100 times with HRP-labeled anti-pig IgA polyclonal antibody (secondary antibody) IgA against PEDV was detected using a detection reagent (trade name: ECL Prime Western Blotting Detection Reagent, manufactured by GE Healthcare, Inc.) using Acris Antibodies.

Based on the results of Western blotting, the presence or absence of induction of IgA production against PEDV was evaluated based on the following evaluation criteria. The results are shown in Table 3 below.
-Evaluation criteria-
"-": No IgA production-inducing activity against PEDV (no Western blot band detected)
"+": IgA production-inducing activity against PEDV (a Western blot band was detected)

  When PEDV (P-5V strain) live vaccine containing adjuvant is orally administered in the first immunization (Example 1), when PEDV not containing adjuvant is orally administered (Comparative Example 1), conventional intramuscular vaccine As compared with the case of administration of No. 2 twice (Comparative Example 2), significant PEDV-specific neutralizing antibody production enhancing activity and PEDV-specific IgA production inducing activity were observed.

(Test Example 2)
MZ0116-2 / 2013 strain (hereinafter referred to as “field strain MZ”), which is a field strain isolated and produced by the following method, after 2 weeks from the booster immunization, in the pigs of Example 1 and Comparative Example 1. ) (10 ^5.0 TCID ₅₀ / mL) 5 mL was orally administered and challenged. In addition, as a control, PED antibody-negative 14-week-old pigs that were not subjected to primary immunization and booster were similarly challenged by oral administration of 5 mL of field strain MZ (10 ^5.0 TCID ₅₀ / mL). Subsequently, observation of clinical symptoms, detection of PEDV RNA in feces, and detection of PEDV RNA in organs were performed by the methods described below.

<< Separation and production of PEDV (MZ0116-2 / 2013 strain (outdoor strain MZ)) >>
A small intestine was collected from a piglet that died of porcine epidemic diarrhea (PED), 0.5 g of this small intestine was placed in a beaded crush tube (manufactured by Bertin Technologies), and a high speed cell crusher (Precellys, Bertin Technologies) After disruption, the suspension was suspended in 5 mL of a culture solution (MEM, manufactured by Thermo Fisher Scientific) and centrifuged. The supernatant after centrifugation was passed through a syringe filter (manufactured by GE Healthcare) having a pore size (pore size) of 0.22 μm to obtain a 10 mass% tissue emulsion. Next, a mixed solution was prepared by mixing 300 μL of the 10 mass% tissue emulsion and 100 μL of 10 μg / mL trypsin-added culture solution. Vero cells previously confluent in a 6-well cell culture plate were inoculated with the entire amount of the mixed solution and cultured at 37 ° C. for 90 minutes. Next, the mixed solution was removed, 4 mL of a 10 μg / mL trypsin-added culture solution was added, and the mixture was cultured at 37 ° C. The cytopathic effect (CPE) was confirmed daily with an optical microscope, and the culture was stopped when CPE was confirmed. Supernatant and cells were collected, frozen and thawed, and then centrifuged and passaged.

<< Identification of MZ0116-2 / 2013 strain >>
In the “identification of the P-5V strain” in Production Example 1, the above-mentioned “identification of the P-5V strain” is used except that the culture supernatant of the field strain MZ was used instead of the culture supernatant of the P-5V strain. A phylogenetic tree analysis was performed in the same manner.
As a result, the strain obtained above was identified as the field strain MZ of PEDV genetic group II (see FIG. 1).

<Observation of clinical symptoms>
In Example 1, Comparative Example 1, and the control, the stool condition and appetite were observed every day from day 0 to day 14 after challenge with the field strain MZ, and evaluated based on the following evaluation criteria. The results of fecal conditions are shown in Table 4 below, and the results of appetite are shown in Table 5 below.
-Evaluation criteria for fecal condition-
"0": Normal stool "1": Soft stool "2": Diarrhea stool "3": Watery diarrhea stool-Evaluation criteria for appetite-
“0”: normal “1”: mild sluggishness “2”: moderate sluggishness “3”: severe sluggishness

  About the state of feces, it was 0 point in all of Example 1, Comparative example 1, and control, and the difference was not recognized (Table 4). On the other hand, the appetite was normal in Example 1, while the appetite was poor in Comparative Example 1 and the control (Table 5).

<Detection of PEDV RNA in rectal swab>
A rectal swab was collected daily using a sterile cotton swab (manufactured by Osaki Medical Co., Ltd.) between day 0 and day 14 after challenge with the field strain MZ in Example 1, Comparative Example 1, and 1 mL of PBS. It was suspended in. This suspension was centrifuged, and viral RNA was extracted using a TRIZol (registered trademark) reagent (manufactured by Thermo Fisher Scientific). PEDV RNA was expressed using SuperScript (registered trademark) III One-Step RT-PCR System (Thermo Fisher Scientific) and PEDV-specific primers (primers 9-F [5'-GATAGTTTTGTAATGTTAGACTACTATAGTGACTACTATAGTGACTACTA ' Amplification was performed with a thermal cycler (Veriti 96-Well Thermal Cycler, manufactured by Thermo Fisher Scientific) by RT-PCR using the primer 9-R [5′-AGCATAGCTAAAGGCATGC-3 ′] represented by SEQ ID NO: 18. The amplified RT-PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and then detected by ultraviolet irradiation. Table 6 below shows the results of calculating the appearance rate (%) of positive individuals in which PEDV RNA was detected with respect to the population of each group.

<Detection of PEDV RNA in organs>
In Example 1, Comparative Example 1, and the control with the field strain MZ on the 14th day after the challenge, the spleen, jejunum, cecum, colon, and mesenteric lymph nodes of each individual were removed, and 10 mass% tissue emulsions were respectively obtained. Produced. PEDV RNA in each organ was extracted from each 10% by mass tissue emulsion using TRIZol (registered trademark) reagent (Thermo Fisher Scientific). Next, with GoTaq (registered trademark) 1-Step RT-qPCR System (manufactured by Promega) and a PEDV-specific primer (primer 10-F [5′-CGCAAAAGACTGAACCCCACTAAC-3 ′] represented by SEQ ID NO: 19) and SEQ ID NO: 20 by primer 10-R [5'-TTGCCTCTGTTGTTACTTGGAGAT- 3 ']) Real-time RT-PCR using represented, RNA mass, StepOnePlus Real-Time PCR System (Thermo Fisher Scientific Inc.) _(log 10 _TCID 50 / mL Equivalent). The results are shown in Table 7 below.

From the results of Table 6, in Comparative Example 1 and the control, the appearance rate of positive individuals in which PEDV RNA was detected was high, whereas in Example 1, positive individuals in which PEDV RNA was detected during the observation period appeared. I didn't. Further, from the results in Table 7, PEDV RNA in the organ was not detected in Example 1 as well.
From these results, it is considered that in Example 1, PEDV was neutralized by secretory IgA secreted from the intestinal mucosa, and protective immunity was established.
In addition, as can be seen in Comparative Example 1 and the control in Table 6, even if PEDV is not detected once, it appears again, and this is known to persist for about 1 to 2 months after infection.

(Example 2)
<First immunization>
The PEDV (P-5V strain) live vaccine 2 produced in Production Example 2 (1 mL, 10 ^7.45 TCID ₅₀ / dose) was administered once intranasally to PED antibody-negative 8-week-old pigs.

<Booster>
Six weeks after the initial immunization, PEDV (P-5V strain) inactivated vaccine 2 (2 mL, 10 ^7.75 TCID ₅₀ / dose) produced in Production Example 4 was administered intramuscularly (injection) once.

(Test Example 3)
For the pig of Example 2, blood was collected immediately before the first immunization and one week after the booster immunization. As a control, blood was collected at 8 and 15 weeks of age for PED antibody-negative 8-week-old pigs that were not subjected to primary immunization or booster immunization. Subsequently, the PEDV neutralizing antibody titer in the collected blood and the PEDV against the PEDV in the same manner as described in the columns of “Measurement of PEDV neutralizing antibody titer” and “Induction of IgA production against PEDV” in Test Example 1 The induction of IgA production was examined. The results are shown in Table 9 below.

In the case of nasal administration of the PEDV (P-5V strain) live vaccine in the first immunization, as in the case of oral administration, the production enhancement activity of the significant PEDV-specific neutralizing antibody and the production induction activity of PEDV-specific IgA are exhibited. Admitted.
Conventionally, there is no report that PEDV infects respiratory organs, and since PEDV infection is limited to the digestive tract, nasal administration induces production of PEDV-specific neutralizing antibodies and PEDV-specific IgA It is an unexpected finding by the present inventors that it can be done.

(Test Example 4)
The pigs of Example 2 were challenged by oral administration of 5 mL of field strain MZ (10 ^5.0 TCID ₅₀ / mL) 2 weeks after the booster immunization. In addition, as a control, 16-week-old pigs negative for PED antibody not subjected to primary immunization and booster were similarly challenged by oral administration of 5 mL of field strain MZ (10 ^5.0 TCID ₅₀ / mL). Subsequently, the detection of PEDV RNA in the rectal swab and the detection of PEDV RNA in the organ were performed in the same manner as described in the column of “Detection of PEDV RNA in rectal swab” in Test Example 2.

<Detection of PEDV RNA in rectal swab>
Table 10 below shows the results of calculating the appearance rate (%) of positive individuals in which PEDV RNA was detected with respect to the population of each group.

<Detection of PEDV RNA in organs>
On day 14 after challenge of Example 2 and the control with the field strain MZ, the jejunum, caecum, colon, and mesenteric lymph nodes of each individual were removed, and PEDV RNA in each organ was determined as “in the organ”. The amount of RNA (equivalent to log ₁₀ TCID ₅₀ / mL) was determined by the same method as that described in the column “Detection of PEDV RNA”. The results are shown in Table 11 below.

(Example 3)
<First immunization>
PEDV (P-5V strain) live vaccine 2 (1 mL, 10 ^7.45 TCID ₅₀ / dose) produced in Production Example 2 was administered once intranasally to PED antibody-negative pregnant pigs.

<Booster>
8 weeks after the initial immunization, PEDV (P-5V strain) inactivated vaccine 2 (2 mL, 10 ^7.75 TCID ₅₀ / dose) produced in Production Example 4 was administered intramuscularly (injection) once.

(Comparative Example 3)
<First immunization>
2 ml of Nisseiken PED live vaccine (manufactured by Nisseiken Co., Ltd.) was intramuscularly administered (injected) once to a pregnant pig negative for PED antibody.

<Booster>
Four weeks after the initial immunization, 2 mL of Nisseiken PED live vaccine (manufactured by Nisseiken) was intramuscularly administered (injected) once.

(Test Example 5)
The piglets born from the pregnant pigs of Example 3 and Comparative Example 3 (2 days after birth) were each challenged by oral administration of 5 mL of the field strain MZ (10 ^5.0 TCID ₅₀ / mL). In addition, as a control, 5 mL of an outdoor strain MZ (10 ^5.0 TCID ₅₀ / mL) was also applied to a piglet (second day after birth) born from a PED antibody-negative pregnant pig that was not subjected to primary immunization or booster immunization. Attacked by oral administration. In addition, even after the piglet attack, the mother pigs of Example 3 and Comparative Example 3 and these piglets were allowed to live together.

<PEDV neutralizing antibody titer in sows>
For Example 3, Comparative Example 3, and control pregnant pigs (mother pigs), blood was collected immediately before the first administration, one week after the additional administration, and one week after the challenge to the piglet. Next, the PEDV neutralizing antibody titer in the collected blood was examined by the same method as described in the column “Measurement of PEDV neutralizing antibody titer” in Test Example 1. The results are shown in Table 13 below.

<IgA production induction for PEDV in sow milk>
In the method described in the column of “Detection of IgA against PEDV by Western blotting” in Test Example 1, the milk of Example 3, Comparative Example 3, and the control mother pig was collected immediately before the attack on the piglet. Except that the blood was changed to the milk, IgA against PEDV in the milk was detected by the same method as in Test Example 1, and the presence or absence of induction of IgA production against PEDV was evaluated by the same method as in Test Example 1. The results are shown in Table 13 below.

<Survival rate of piglets>
The piglets born from Example 3, Comparative Example 3, and control pregnant pigs were confirmed daily on the 0th to 7th day after challenge with the field strain MZ. The results are shown in Table 14 below.

<Histopathological observation of the small intestine of piglets>
The small intestine of each piglet extracted at the time of “detection of PEDV RNA in the piglet organ” was formalin-fixed, and paraffin sections were prepared by a conventional method. Next, paraffin was removed from the paraffin section using xylene. Next, for rehydration of the sections, the sections were immersed in an aqueous ethanol solution having a concentration gradient, the sections were rehydrated, and then ethanol was removed using distilled water. The slice specimen from which ethanol was removed was immersed in a hematoxylin solution for 20 minutes, washed with distilled water, and then immersed in an eosin solution for 2 minutes for staining. Next, after dehydrating by immersing in an ethanol aqueous solution having a concentration gradient, the tissue specimen was immersed in xylene and penetrated to obtain a tissue specimen. The prepared tissue specimen was observed with an optical microscope, and a comprehensive evaluation on villous atrophy, villous cell bombardment, villi flattening, and inflammation of the villous basement membrane was performed based on the following evaluation criteria. Since several piglets were born from one mother pig, the score of the evaluation criteria was calculated as the average value of the piglets in each group of Example 3, Comparative Example 3, and the control, and Student's T test The significant difference was confirmed. The results are shown in Table 15 below.
-Evaluation criteria-
“0”: Normal “1”: Mild “2”: Moderate “3”: Severe

Examples of the aspect of the present invention include the following aspects.
<1> a first administration step in which a swine epidemic diarrhea virus live vaccine and an adjuvant are administered to pigs by either oral administration or nasal administration;
A method for preventing or treating porcine epidemic diarrhea, comprising: a second administration step of administering the inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant to the pig by intramuscular administration.
<2> The method for preventing or treating swine epidemic diarrhea according to <1>, wherein the adjuvant is a microemulsion adjuvant.
<3> Porcine epidemic diarrhea virus live vaccine for oral or nasal administration, adjuvant for oral or nasal administration, inactivated vaccine of porcine epidemic diarrhea virus for intramuscular administration, and adjuvant for intramuscular administration It is a vaccine kit characterized by having.
<4> The vaccine kit according to <3>, wherein the adjuvant for oral or nasal administration and the adjuvant for intramuscular administration are microemulsion adjuvants.
<5> It is used for pigs given booster immunity after intramuscular administration of an inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant,
It is a vaccine for oral or nasal administration, characterized in that it comprises a live vaccine of the porcine epidemic diarrhea virus and an adjuvant.
<6> The vaccine for oral or nasal administration according to <5>, wherein the adjuvant is a microemulsion adjuvant.
<7> A swine epidemic diarrhea virus live vaccine and adjuvant are used orally or nasally administered to pigs given initial immunity,
A vaccine for intramuscular administration, comprising the inactivated vaccine of porcine epidemic diarrhea virus and an adjuvant.
<8> The vaccine for intramuscular administration according to <7>, wherein the adjuvant is a microemulsion adjuvant.

  The method for preventing or treating swine epidemic diarrhea according to the present invention includes a vaccine kit, a vaccine for oral or nasal administration, and a vaccine for intramuscular administration, the activity of inducing production of neutralizing antibodies specific for swine epidemic diarrhea virus, and Since it has excellent humoral immune response-inducing activity, it can be suitably used for the prevention or treatment of swine epidemic diarrhea.

Claims (4)

A first administration step of administering a porcine epidemic diarrhea virus live vaccine and a microemulsion adjuvant to a pig by either oral or nasal administration;
A second administration step of administering the inactivated vaccine of porcine epidemic diarrhea virus and a microemulsion adjuvant to the pig by intramuscular administration, and prevention of porcine epidemic diarrhea targeting pigs Or a treatment method. Porcine epidemic diarrhea virus live vaccine for oral or nasal administration, microemulsion adjuvant for oral or nasal administration, inactivated vaccine of porcine epidemic diarrhea virus for intramuscular administration, and microemulsion adjuvant for intramuscular administration A vaccine kit characterized by comprising: Used for pigs given booster immunization after intramuscular administration of an inactivated vaccine of porcine epidemic diarrhea virus and microemulsion adjuvant,
A vaccine for oral or nasal administration, comprising a live vaccine of the porcine epidemic diarrhea virus and a microemulsion adjuvant. It is used for pigs given primary immunity by oral or nasal administration of live porcine epidemic diarrhea virus vaccine and microemulsion adjuvant,
A vaccine for intramuscular administration, comprising the inactivated vaccine of porcine epidemic diarrhea virus and a microemulsion adjuvant.